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Depletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date. We have generated a T cell receptor mimic antibody, “Foxp3-#32,” recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs.

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

Purpose: The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide–MHC complexes can be targets for CAR T-cell therapy against solid tumors. Experimental Design: We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158–166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. Results: We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). Conclusions: This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478–88. ©2016 AACR.

Hepatocellular carcinoma (HCC) is the 5th most prevalent and 3rd most lethal cancer worldwide, due to limited treatment options. The lack of tumor-specific membrane targets is one of the key challenges currently facing immunotherapy for solid tumors, where on-target/off-tumor activity can lead to severe adverse responses. Most HCCs overexpress Alpha-Fetoprotein (AFP), a secreted fetal glycoprotein rarely expressed in adult tissues, making AFP an ideal target for immunotherapy. However, since AFP is only expressed intracellularly and secreted, it cannot be targeted with traditional antibodies against cell-surface antigens. To overcome this limitation, we exploited the fact that intracellular antigens, including AFP, are processed into peptides and presented by class I major histocompatibility complexes (MHCs) on the surface of tumor cells. Using an antibody discovery platform optimized for identifying candidates that bind peptide/MHC complexes, we developed ET1402L1, a fully-human scFv specifically targeting an immunogenic AFP peptide complexed with human leukocyte antigen (HLA)-A*02:01. ET1402L1 binds HLA-A*02:01/AFP with exquisite specificity and does not cross-react with naked HLA-A*02:01 molecules or with HLA-A*02:01 complexed with a panel of control peptides from endogenous human proteins. T cells engineered to express a second generation chimeric antigen receptor (CAR) constructed with ET1402L1 responded to HLA-A*02:01/AFP-expressing cells by degranulating and releasing IFN-γ, IL-2, IL-6, IL-8, IL-10, GM-CSF, and TNFα in an antigen-selective manner. In a cell killing assay, ET1402L1-CART selectively lysed HCC cells that expressed both HLA-A*02:01 and AFP, while sparing cells from multiple tissue types that were negative for either. In vivo, ET1402L1-CART demonstrated potent anti-tumor activity in multiple HCC xenograft models. While intravenous administration of ET1402L1-CART significantly inhibited the growth of established subcutaneous (s.c.) Hep G2 tumors (an HCC cell line positive for HLA-A*02:01 and AFP), intratumoral delivery resulted in rapid and prolonged tumor regression with complete regression observed in 75% of animals. These effects were recapitulated in a s.c. tumor model using another liver-derived HLA-A*02:01-positive cell line (SK-HEP-1) engineered to express the AFP peptide. Lastly, intraperitoneal injection of ET1402L1-CART led to regression of tumors in a disseminated abdominal HCC xenograft model. Our data demonstrates that CAR-T cell therapy targeting intracellular antigens via peptide/MHC complexes can effectively eradicate solid tumors in vivo. This approach expands the spectrum of antigens available for redirected T cell therapy against solid malignancies and provides a promising future therapy for HCC. A phase I clinical trial testing ET1402L1-CART in HCC patients is expected to begin in 2016. Citation Format: Hong Liu, Yiyang Xu, Jingyi Xiang, Li Long, Shon Green, Zhiyuan Yang, Jingwei Lu, Neal Cheng, Lucas H. Horan, Bin Liu, Su Yan, Pei Wang, Juan Diaz, Lian-xing Liu, Vivien Chan, Cheng Liu. ET1402L1-CART, a T cell therapy targeting the intracellular tumor antigen AFP, demonstrates potent antitumor activity in hepatocellular carcinoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2299.